Macrophage IL-1β mediates atrial fibrillation risk in diabetic mice

Diabetes mellitus (DM) is an independent risk factor for atrial fibrillation (AF). The mechanisms underlying DM-associated AF are unclear. AF and DM are both related to inflammation. We investigated whether DM-associated inflammation contributed to AF risk. Mice were fed with high-fat diet to induce type II DM and were subjected to IL-1β antibodies, macrophage depletion by clodronate liposomes, a mitochondrial antioxidant (mitoTEMPO), or a cardiac ryanodine receptor 2 (RyR2) stabilizer (S107). All tests were performed at 36–38 weeks of age. DM mice presented with increased AF inducibility, enhanced mitochondrial reactive oxygen species (mitoROS) generation, and activated innate immunity in the atria, as evidenced by enhanced monocyte chemoattractant protein-1 (MCP-1) expression, macrophage infiltration, and IL-1β levels. Signs of aberrant RyR2 Ca2+ leak were observed in the atria of DM mice. IL-1β neutralization, macrophage depletion, and exposure to mitoTEMPO and S107 significantly ameliorated the AF vulnerability in DM mice. Atrial overexpression of MCP-1 increased AF occurrence in normal mice through the same mechanistic signaling cascade as observed in DM mice. In conclusion, macrophage-mediated IL-1β contributed to DM-associated AF risk through mitoROS modulation of RyR2 Ca2+ leak.


INTRODUCTION
Type II diabetes mellitus (DM) is one of the most common chronic diseases in the world (1).
DM is also an independent risk factor for the development of atrial fibrillation (AF) (2,3), increasing the risk of developing AF by 40% (4).AF is the most prevalent human arrhythmia (5) and an estimated 2.5% of patients with AF have diabetes (4).The presence of comorbid DM and AF increases the risks of major cardio-cerebrovascular events and all-cause mortality compared to either condition alone (6).DM causes electrical, structural, and autonomic remodeling in atria leading to the development of AF (7).Nevertheless, the exact cellular and molecular mechanisms inciting and maintaining DM-associated AF and diabetic atrial remodeling are not fully understood.
Recently, we have reported that DM activates inflammatory macrophages to secrete IL-1β, resulting in overproduction of mitochondrial reactive oxygen species (mitoROS) in ventricle (8,9).MitoROS contributes to DM-associated ventricular arrhythmic risk and heart failure with preserved ejection fraction (HFpEF) through oxidizing the downstream ryanodine receptor 2 (RyR2) channel and cardiac myosin binding protein C, respectively (9,10).Considering the strong epidemiological interlink among DM, AF and HFpEF, it is plausible that there may be common mechanistic signals underlying DM-associated cardiac electrical and functional abnormalities.
Therefore, in this study, we investigated whether macrophage-mediated IL-1β contributed to DM-associated AF via redox modulation of diastolic Ca 2+ release.

AF inducibility was increased in DM mice
Type II DM was induced by feeding mice with high fat diet (HFD) for at least 30 weeks.This HFD-induced DM model has been demonstrated and characterized in our previous studies (8,10).

Innate immunity was activated in DM atria
To investigate whether innate immunity was activated in DM atria, we first compared the atrial level of monocyte chemoattractant protein-1 (MCP-1), a key chemokine that regulates macrophage migration and infiltration (11).As shown in Figure 2A, the atrial MCP-1 level was significantly higher in DM mice, compared to that of control mice (1.02 ± 0.04 in control vs. 1.17 ± 0.05 in DM, P=0.045).An elevation of MCP-1 promotes macrophage infiltration (12).As expected, an enhanced expression of the macrophage marker CD68 was observed in DM mouse atria (Figure 2B, 0.86 ± 0.06 in control vs. 1.18 ± 0.07 in DM, P=0.011), indicating increased macrophage infiltration in DM atria (Supplemental Figure S2).Activated macrophages are important sources of a potent inflammatory cytokine, IL-1β.In our study, we found that the IL-1β level was much higher in DM atria (Figure 2C, 2.83 ± 0.74) than control (0.91 ± 0.07, P=0.041).
These results suggested that macrophage-mediated inflammation was enhanced in DM mouse atria.

Macrophage-mediated IL-1β contributed to DM-associated AF
We have previously reported that macrophage-mediated IL-1β contributed to DM-associated diastolic dysfunction (DD) (9).DD shares some common risk factors with AF, such as DM, hypertension, obesity, et al (13).To investigate whether a similar mechanism contributes to DMassociated AF, we treated DM mice with either Clodronate liposomes or IL-1β neutralizing antibodies for 2 weeks.We have demonstrated in our previous publication that a 2-week treatment of Clodronate liposomes reduces the number of cardiac macrophages by 50% (9).In this study, we found that macrophage depletion remarkably reduced the IL-1β level in DM atria (Figure 3A

A mitochondria-targeted antioxidant eliminated AF risk in DM mice
AF is related to increased oxidative stress (14), and oxidative stress is considered a central mediator of AF (15).Mitochondria are the major reactive oxygen species (ROS) source in cardiomyocytes (CMs) (16) and mitoROS promotes AF (17) .We have previously reported increased mitoROS level in the ventricular CMs of DM mice and that IL-1β raises the mitoROS level of ventricular CMs in a dose-dependent response (8).Similar to DM ventricles, we found that the mitoROS level was more than doubled in DM atrial CMs (Figure 4A and 4B, 100 ± 12% in control vs. 266 ± 14% in DM, P<0.0001).A mitochondrial specific antioxidant, mitoTEMPO, was administered to DM mice for 2 weeks.At the end of the treatment, the atrial CM mitoROS level was significantly lowered (Figure 4A and 4B, 138 ± 15%, P<0.0001 vs. DM), and the AF inducibility was completely inhibited by mitoTEMPO (Figure 1G, P=0.005).In addition, mitoTEMPO reduced CD68 expression in DM atria (Figure 4C, 0.98 ± 0.25 in DM vs. 0.27 ± 0.02 in DM+mitoTEMPO, P=0.048), suggesting reduced macrophage infiltration by inhibiting mitoROS.These data implied the involvement of atrial mitoROS in DM-associated AF risk and a positive feedback loop between mitoROS and atrial macrophage infiltration.
These results indicated that atrial MCP-1 overexpression increased AF vulnerability, presumably via inducing macrophage infiltration.
Moreover, a similar signaling cascade as observed in DM atria was present in MCP-1 OE mice.The mitoROS level was elevated by 1.5-fold in atrial cardiomyocytes from MCP-1 OE mice (Figure 7A, P=0.0008) compared with the AAV9-plain vector treated mice.Both oxi-CaMKII and phosphorylated RyR2 (Ser2814) were significantly higher in MCP-1 OE atria than in control.The oxi-CaMKII increased from 1.21 ± 0.12 in control to 1.94 ± 0.10 in MCP-1 OE mice (Figure 7B and 7D, P=0.002) and p-RyR2 (Ser2814) increased from 0.73 ± 0.12 in control to 2.60 ± 0.79 in MCP-1 OE mice (Figure 7C and 7D, P=0.047).These data suggested that, similar to DMassociated AF, mitoROS-mediated RyR2 modification linked the inflammation and AF in MCP-1 OE mice.

DISCUSSION
In this study, we found that DM caused AF vulnerability accompanied with enhanced atrial MCP-1 and IL-1β levels, increased atrial macrophage infiltration, elevated mitoROS production, and RyR2 phosphorylation in atrial CMs.Neutralizing IL-1β, depleting macrophages, scavenging mitoROS, or blocking Ca 2+ leak from RyR2 channels, improved AF vulnerability in DM mice.
The above signaling cascade was further confirmed in the atrial specific MCP-1 overexpressing mice in which AF risk was significantly enhanced.Taken together, these findings indicated that activated innate immunity contributed to the DM-associated AF tendency via IL-1β-mediated atrial electrical remodeling of RyR2 through mitoROS modulation.
Both DM and AF are associated with inflammation (22)(23)(24)(25).DM is well known as a chronic inflammatory disease.Activation of the innate immune response is closely involved in the pathogenesis of type II DM (26).Macrophages are a major component of innate immunity and the major immune cell population in hearts (27,28).Type II DM patients present with higher plasma MCP-1 levels and increased CD68 + macrophages in the atrial myocardium when compared to non-DM patients (29,30).Macrophages can adopt pro-inflammatory or anti-inflammatory phenotype, and DM favors the pro-inflammatory macrophages (31).
There is considerable evidence to suggest that macrophages can contribute to AF (32).The main population of immune cells in human left atrial appendages of patients with AF are active monocytes/macrophages (27,33).Increased MCP-1 and pro-inflammatory macrophage infiltration in atria have been reported in both human and animals with AF (33)(34)(35)(36)(37)(38)(39).Blocking monocyte recruitment reduces atrial macrophage infiltration and lowers the incidence of hypertension induced AF (40).Increased macrophage pro-inflammatory polarization (Inos + and Arg1 -) is found in the mouse and canine atria after lipopolysaccharide (LPS) induced AF (41).We and others have reported a similar role of macrophages on the diabetic ventricular arrhythmic risk (8,42).Therefore, macrophage-mediated inflammation may be a key link between DM and arrhythmia.
In the current study, we found enhanced MCP-1 and macrophage infiltration in the DM mouse atria (Figure 2A and 2B, Supplemental Figure S2).The increased IL-1β in diabetic atria (Figure 2C) and the upregulated gene expression of CCR2, a pro-inflammatory macrophage marker, in MCP-1 overexpressing atria (Figure 6D) supports that the accumulated macrophages in DM atria were pro-inflammatory.That is consistent with our previous report of an increased shift toward pro-inflammatory macrophages in the ventricles of the same DM mouse model (9).Moreover, we observed that depleting macrophages attenuated DM-associated AF vulnerability (Figure 3B), and atrial specific overexpression of MCP-1 induced AF in normal mice (Figure 6E), suggesting the central contribution of pro-inflammatory macrophages in DM-associated AF.
Although macrophage depletion was sufficient to reduce arrhythmic risk, we cannot rule out a role for other inflammatory cell types in DM-associated AF.In another report, neutrophils are a main ROS source and play a pro-fibrotic role in AF genesis, and T cells and B cells contribute to AF via regulating innate immunity and producing autoantibodies respectively (27).Those three leukocyte subsets have been found to be involved in diabetic cardiomyopathy (31,43).
Pro-inflammatory macrophages secrete inflammatory cytokines such as IL-1β, IL-6, and TNFα, all of which are elevated in AF patients or associated with the outcome of AF (44)(45)(46)(47)(48)(49).IL-1β is an independent risk factor for persistent AF in patients after coronary artery bypass grafting surgery (45).AF is remarkably associated with elevated IL-6 in the patients with coronary artery disease, chronic obstructive pulmonary disease, chronic kidney diseases, and many other systemic inflammatory diseases (49)(50)(51).High levels of TNF-α are reported in patients with valvular AF (52).In LPS-induced AF, pro-inflammatory macrophages induce atrial electrical remodeling through IL-1β and TNF-α (41).In our DM mouse model, we showed that DM-associated AF was at least partially mediated by macrophage secreted IL-1β as evidenced by the efficacy of macrophage depletion and IL-1β neutralization in blocking AF (Figure 1G and Figure 3B).Nevertheless, we did not rule out the role of other cytokines in DM-associated AF.Further, we did not examine other cell sources of IL-1β.For example, cardiomyocytes can also release inflammatory cytokines through the activation of NLRP3 (NACHT, LRR, and PYD domain containing protein 3) inflammasome that may contribute to the proclivity for AF (53).Nevertheless, in the present study, macrophage depletion normalized atrial IL-1β level (Figure 3A), establishing macrophages as the main source of IL-1β in DM atria.
MitoROS can cause AF through promotion of SR Ca 2+ leak via RyR2 oxidation or CaMKIImediated phosphorylation (17,59,60).CaMKII can be activated by mitoROS via oxidation (15,61,62), and oxidized CaMKII (oxi-CaMKII) is elevated in AF atria (63).We have similar observations that DM elevated mitoROS production and CaMKII oxidation in atria and led to increased RyR2 phosphorylation (Figure 4 and 5).A mitochondrial antioxidant protected DM mice from inducible AF (Figure 1G), providing additional proof of a redox mechanism in DMassociated AF.In addition to redox modulate intracellular Ca 2+ homeostasis, mitoROS can also promote AF through perpetuating inflammation via activating the NLRP3 inflammasome and driving inflammatory cytokine release, such as IL-1β and IL-18 (64).This is reflected in our finding that scavenging mitoROS inhibited macrophage infiltration (Figure 4C), a proof of a vicious positive feedback loop between mitoROS and macrophage-mediated inflammation in the development of DM-associated AF.
The suppression of DM-associated AF risk by RyR2 stabilization (S107) indicated the contribution of RyR2 channels to AF in DM, potentially by oxi-CaMKII-mediated phosphorylation (Figure 5).Similarly, Mesubi et al. reported that diabetic AF is dependent on oxi-CaMKII activated RyR2 Ca 2+ leak (65).Nevertheless, we cannot rule out the possibility of direct RyR2 oxidation by ROS leading to the pathogenesis of AF, as reported by Xie et al (17).
IL-1β can also contribute to AF by affecting other cardiac ion channels or atrial fibrosis (41,46,66).In LPS-induced AF, IL-1β contributes to the atrial electrical remodeling by leading to downregulated L-type calcium channel currents and decreased atrial effective refractory period (41).Inhibiting IL-1β-induced atrial fibrosis prevents post-operative AF (66).In our DM mouse model, atrial fibrosis was not a potent contributor to AF since atrial collagen level was not altered (Figure 1F and Supplemental Figure 1).Instead, our data suggest RyR2-mediated Ca 2+ leak as a potential trigger mechanism for AF initiation (Figure 5).Nevertheless, the roles of other electrical remodeling in DM-associated AF were not examined and need further clarification.
Previously, we have investigated the inflammatory mechanism in DM-associated ventricular arrhythmic risk and diastolic dysfunction (8,9).In the present study, we found that DM-associated AF shared a common mechanistic signaling cascade with diabetic diastolic dysfunction as well as ventricular arrhythmic risk, namely MCP-1 elevation, macrophage infiltration, IL-1β secretion, mitoROS overproduction, and post-translational modification of target proteins.This finding is consistent with the epidemiological association of AF and diastolic heart failure (67).A recent randomized double-blind placebo-controlled clinical trial (the CANTOS trial) showed antiinflammatory therapy with an IL-1β specific antibody (canakinumab) substantially reduced cardiovascular events (68), supporting our conclusion that IL-1β is a key signaling component in DM-associated cardiovascular complications.
It must be recognized that mice may not be an ideal model of human electrophysiological diseases.Mouse cardiac physiology differs from human in aspects such as heart size, basal heart rate, action potential duration, ionic currents for cardiomyocyte repolarization (69).Thus, extrapolating mouse data for human electrophysiological implications needs to be done cautiously.
In summary, DM results in activation of a cardiac innate immune response associated with increased AF risk.AF vulnerability could be ameliorated by depleting macrophages, antagonizing IL-1β, scavenging mitoROS, or inhibiting SR Ca 2+ leak.Each of these approaches represents a possible new therapy for preventing DM-associated AF risk.

Sex as a biological variable.
Our study only examined male diabetic mice as female C57BL/6J mice were less susceptible from HFD-induced DM (70).For atrial MCP-1 overexpressing study, both male and female mice were used, and similar findings were observed in both sexes.
A group of normal C57BL/6J mice (male and female, purchased from Jackson Laboratory, Bar Harbor, ME) were intravenously injected with adeno-associated viral vector (serotype 9, AAV9) (5×10 11 genome copies/mouse) at the age of 9 weeks.The AAV9 vector (VectorBuilder Inc., Chicago, IL) is driven by atrial natriuretic factor (ANF) promoter to overexpress MCP-1 specifically in atrial CMs.The sex-and age-matched control mice were injected with the same copy number of AAV9-EGFP control virus.
All subsequent tests were performed at 36-38 weeks of age or at one month post virus injection.

Echocardiographic evaluation of cardiac function
Echocardiography was performed using the Vevo 2100 (VisualSonics, Toronto, Canada) ultrasound system as in previous studies (9).Mice were anesthetized with 1-2% isoflurane in oxygen at 1 L/min with the body temperature and the heart rate maintained at 37-38 °C and above 400 bpm, respectively, during the scanning.B-mode images along the left ventricular parasternal long axis and then M-mode images at the mid-papillary level were obtained to calculate ejection fraction and chamber size.E/E' was assessed in the subcostal 4-chamber view by pulsed-wave and tissue Doppler imaging to evaluate diastolic function.Measurements were averaged from five consecutive beats during expiration.

Programmed intracardiac stimulation
Programmed intracardiac stimulation was performed to assess AF inducibility as described previously (72).A standard limb electrocardiogram (ECG) was recorded from subcutaneously inserted needle electrodes.Atrial and ventricular intracardiac electrograms were recorded using a 1.1F Millar electrophysiology catheter (Millar Instruments, Houston, TX) advanced through the right jugular vein into the right ventricle.Surface and intracardiac electrophysiology parameters were recorded at sampling rate of 4000 Hz.Right atrial pacing was performed using 2-ms current pulses delivered by an external stimulator (STG2004, Multi Channel Systems, Reutlingen, Germany) along with MCStimulus software (Multichannel System, Baden-Württemberg, Germany).AF was induced by an overdrive pacing protocol, starting with 2-second burst pacing at a cycle length of 40 ms and decreasing in each successive burst by a 2-ms decrement to a cycle length of 10 ms.Inducible AF was defined the occurrence of rapid, fragmented atrial electrograms with irregular R-R intervals lasting at least 1 second.To determine whether AF inducibility was reproducible, mice were subjected to the same atrial burst-pacing protocols 3 times, and only the mice that exhibited ≥ 2 times of evoked AF by pacing were considered AFpositive.The programmed pacing was performed in a blinded manner.

Quantitative Real-time PCR (qPCR)
Total RNA was extracted from the left and right atrial tissues utilizing the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions.The purity and concentration of the isolated RNA were assessed spectrophotometrically.Subsequent to extraction, reverse transcription was conducted to synthesize complementary DNA (cDNA) from the total RNA using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific), following the protocol provided by the manufacturer.Quantitative Polymerase Chain Reaction assays were performed to evaluate the expression levels of specific genes.These assays were carried out using the PowerUp SYBR Green Master Mix (Applied Biosystems) on a 7500Fast Real-Time PCR System (Applied Biosystems).Specific primer sets were employed to amplify target gene sequences (mouse Ccr2 forward primer -GCTGTGTTTGCCTCTCTACCAG, reverse primer -CAAGTAGAGGCAGGATCAGGCT; mouse Gapdh forward primer -CTTCAACAGCAACTCCCACTCTT, reverse primer -TGTCATACCAGGAAATGAGCTTGA).The relative gene expression levels were calculated using the 2 -ΔCt method, with normalization to endogenous control gene expression to account for variability in cDNA input levels.
Cardiomyocytes were separated from interstitial cells by settling for 10 min.The cell pellet was then collected for mitoROS measurement.

MitoSOX Red staining and mitoROS measurement
MitoROS was measured in the isolated atrial cardiomyocytes by an inverted confocal laser scanning microscope (Olympus Life Science Solutions Americas Corp., Waltham, MA) as described previously (73).Briefly, isolated cardiomyocytes were resuspended in standard